International Journal of Environment, Agriculture and Biotechnology (IJEAB)
http://dx.doi.org/10.22161/ijeab/1.4.48
Vol-1, Issue-4, Nov-Dec- 2016
ISSN: 2456-1878
Cultural and morphological variations of
Colletotrichum spp associated with anthracnose of
various fruits in Cameroon
*Keuete Kamdoum E.1, Tsopmbeng Noumbo G.R.1, Kuiate J.R.2
1
Laboratory of Applied Botany, Faculty of Science, University of Dschang, Box 67 Dschang, Cameroon
Laboratory of Microbiology and Antimicrobial Substances, Faculty of Science, University of Dschang, Box 67 Dschang, Cameroon
2
Abstract— The anthracnose of fruits due to Colletotrichum
spp. is one of the principal fungal diseases which affects the
production and marketing of fruits in Cameroon. Isolates of
Colletotrichum were collected from various fruits and
characterised for cultural and morphological variations. The
results show that Colletotrichum colonies varied in the
appearance of their culture ranging from fibrous, compact and
cottony colonies. The colour of colonies ranged between
whitish to greyish, pinkish and greyish green. AVIS1, BAIS2,
MAIS1, PAIS1 and PLIS1 had an intermediate growth varying
between 13.02 to 13.61 mm/day, whitish to greyish mycelium
and fusiform conidia of size ranging between 19.98 x 4.17 and
21.29 x 5.14 µm. BAIS2 had the fastest growth (17.19 mm/day)
with a pinkish fibrous mycelium and cylindrical or spindleshaped conidia of 25.62 x 6.04 µm in size and a sporulation
rate of 8.69 x 104. These results highlight some variations in
morphocultural characteristics of Colletotrichum species,
however molecular analyses are still going on for adequate
differentiation among those isolatesfrom different fruits.
Keywords—Anthracnose, Colletotrichum spp., fruits,
morphocultural, variations.
I.
INTRODUCTION
Anthracnose of fruits caused by the Colletotrichum species is
one of the most important postharvest diseases of fruits.
Symptoms of anthracnose include black and sunken lesions
with spore masses or acervuli in the lesion. Infection on fruits
usually starts during the development of the fruit but remains
quiescent until the fruit ripens; symptoms often manifest
during storage and marketing (Prusky and Plumbley 1992).
Anthracnose becomes severe when the fruits are wounded by
scratches during handling and transportation, making the fruit
unmarketable. Two types of symptoms are found on fruits. The
commonest is a dark-brown lesion which is slightly sunken
with raised rims (Bailey et al., 1992; Agrios, 2005). This can
be found on very young fruits or matured fruits in storage or
transit. The lesions can enlarge on the fruit surface and
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eventually penetrate the fruit and infected young fruits usually
drop (Nelson, 2008). The black necrotic lesions may or may
not be accompanied by bright orange acervuli which are the
fruiting bodies of the pathogen (Agrios, 2005). The second
type of symptom is commonly referred to as tear strain
symptom in which are linear necrotic regions on the fruit that
may or may not be associated with superficial cracking of the
fruit epidermis causing an alligator skin effect on the fruit
surface (Nelson, 2008). According to Dodd et al., (1992)
anthracnose causes premature fruit drop and direct reduction in
quality of ripe fruits and shortening storage life time.
The disease may develop on fruits belonging to extremely
varied families. For a good number of fruits, it represents the
principal post-harvest disease of fungal origin as it is the case
in mango, plums, pawpaw, banana and avocado (Sangeetha
and Rawal, 2009; Hala and Coulibaly, 2006; COLEACP,
2011). Contamination on fruits causes necrosis and in long
term, putrefaction (Prusky et al., 2000). This degrades the
quality of fruit and post-harvest losses of up to 100% of the
production can be recorded (COLEACP, 2008). The pathogen
can infect young fruits at fruit initiation (Arauz, 2000;
COLEACP, 2008) or on already developed fruits. The most
significant damages on the fruits are generally expressed after
harvest (Sanders and Korsten, 2003).
Differentiation between Colletotrichum species based on host
range or host of origin may not be a reliable criterion for fungi
of this genus, since taxa such as C. gloeosporioides, C.
dematium, C. acutatum, and others infect a broad range of host
plants. Some taxa appear to be restricted to host families, genus
or species within those families, or even cultivars, whereas
others have more extensive host ranges (Freeman et al., 1998).
Identification of Colletotrichum spp. is therefore a fundamental
criterion in the development of more control measures.
Traditional
identification
and
characterisation
of
Colletotrichum species has relied primarily on differences in
morphological features such as colony colour, size and shape
of conidia and appressoria, growth rate, presence or absence of
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Vol-1, Issue-4, Nov-Dec- 2016
ISSN: 2456-1878
setae, and existence of the Glomerella teleomorph (Smith and
Black, 1990; Gunnell and Gubler, 1992; Sutton, 1992). Studies
of these features on Colletotrichum species have not yet been
conducted in Cameroon. The present preliminary are aimed at
investigating on the morphocultural variation of the genus
Colletotrichum isolated from five postharvest fruits sold in
market in Cameroon.
sodium hypochlorite solution for 5 minutes, followed by 3
changes with washings with sterilized distilled water. The
surface sterilized diseased pieces were then aseptically
transferred separately to Petri dishes containing 20 ml Potato
Dextrose Agar (PDA) medium amended with Chloramphenicol
(1 g/l) to prevent bacterial contamination and then incubated at
24 ± 2° C. After 2 to 3 days of incubation, the growing
mycelium was sub-cultured on fresh PDA medium until pure
cultures. In this way, the cultures of different isolates were
II.
MATERIALS AND METHODS
obtained and maintained in a refrigerator at 4° C.
Isolation of Colletotrichum species from infected fruits
Pulp fragment from each fruit (avocado, banana, mango,
Morphological identification of Colletotrichum isolates was
pawpaw and plum) showing typical anthracnose symptom
carried out based on the cultural characteristics and with the
were thoroughly washed in tap water and separately cut into
help of identification keys of mycology (Barnet and Hunter,
small pieces at about half a centimeter in size, showing half
1972; Cannon et al., 2008; Prihastuti et al., 2009; Phoulivong
et al., 2010; Su et al., 2011). A summary of Colletotrichum
healthy and half diseased tissue, with the help of previously
isolates used in this study are listed in Table 1.
sterilized blade. The pieces were surface sterilized with 5 %
Table.1: Origin of Colletotrichum spp isolates used for the study.
Isolate code
Host fruit
Scientific Names of fruit
Avocado
Persea americana
Avocado
Persea americana
Banana
Musa sapientum
Banana
Musa sapientum
Mango
Mangifera indica
MAIS2
Mango
Mangifera indica
PAIS1
Pawpaw
Carica papaya
PLIS1
Plum
Dacryodes edulis
AVIS1
AVIS2
BAIS1
BAIS2
MAIS1
Cultural characteristics
Mycelial discs (6 mm) of 7 day old culture of Colletotrichum
isolates were transferred aseptically to the center of PDA plates
and incubated at 24 ± 2° C. Culture characteristics such as
colony aspect and color were observed and recorded after 7
days of incubation. Colony diameter was measured in two
perpendicular directions on the reverse side Petri dishesevery
two days after incubation and growth rate was calculated on
day 7 followed the formula of Sofi et al. (2013).
Growth rate =
This experiment was repeated four times.
Morphological characteristics
For each isolate, a conidial suspension was prepared by
carefully brushing 10 days old cultures into 20 ml of sterilized
distilled waterin a 90 mm Petri dish and a drop of Tween 20
was added to each plate to homogenise the suspension.
Conidial suspensions obtained were filtered through a double
layer of cheesecloth to remove leaf debris. Then a drop of
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conidial suspension from each isolate of Colletotrichum from
different fruits was mounted and quantified using a
haematocymeter. Afterwards, the sizes of conidia were
determined by measuring 50 random conidia with a calibrated
microscope (Olympus brand) at magnification 400X. The
shapes of these conidia were also recorded. The experiment
was repeated fourtimes.
Experimental Design and Statistical Analysis
All the experiments were conducted following a completely
randomized design (CRD), and data on radial growth, growth
rate and sporulation rate were analyzed using an analysis of
variance (ANOVA) in SPSS software version 20.0 and mean
separated with Duncan’s Multiple Range test (DMR) at a 5 %
probability level.
III.
RESULTS
Cultural characteristics
On the basis culturalcharacteristics, isolates from fruits showed
different colony aspect and colourafter 7 days (Fig 1). Colonies
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produced by AVIS1, MAIS1, PAIS1 and PLIS1 collected
respectively from avocado, mango, papaw and plums fruits
varied from whitish to greyish with cottony aerial mycelium
and a few bright orange masses near the inoculum point.
BAIS1 and MAIS2 isolates from banana and mango
Vol-1, Issue-4, Nov-Dec- 2016
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producedgreyish green colonies with compact mycelium.
MAIS2 presented sparse white fluffy aerial mycelium.
Colonies produced by BAIS2 from banana with pinkish
colouration had showed fibrous mycelia. AVIS2 isolates from
avocado produced greyish colonies with fibrous mycelia.
Fig.1: Colonies of Colletotrichum species on PDA. (a) pure culture of AVIS1; (b) pure culture of AVIS2; (c) pure culture of BAIS1; (d)
pure culture of BAIS2; (e) pure culture of MAIS1; (f) pure culture of MAIS2; (g) pure culture of PAIS1 and (h) pure culture of PLIS1.
BAIS2 isolate obtained from banana, had a highest radial
growth of isolates ranged from 7.37 to 17.19 mm/day. The rate
growth of 79.32 mm, followed by AVIS1, BAIS1, MAIS1,
of growth of isolate BAIS2 (17.19 mm/day) was the fastest,
PAIS1 and PLIS1 that had average radial growths ranging
followed by AVIS1, BAIS1, MAIS1, PAIS1 and PLIS1 (13.41
from 54.05 to 65.27 mm while AVIS2 and MAIS2 had lowest
to 13.61 mm/day). The slowest growing culture was isolated
radial growth of 38.47 to 40.06 mm (Table 2). Also, the rate of
MAI2 and AVIS2 (7.37 and 7. 62 mm/day).
Table.2: Cultural characteristics of Colletotrichum isolates from different fruits
Growth rate day 7
Isolates
Aspect of
Colour of colony
Radial growth on the
(mm/day)
code
colony
7th day (mm)
Cottony
Whitish to greyish
61.54 ± 2.25b
13.02 ± 0.89b
AVIS1
AVIS2
Fibrous
Greyish
38.47 ± 3.15c
7.62 ± 0.74c
BAIS1
Compact
Greyish green
63.81 ± 2.57b
13.61 ± 0.72b
BAIS2
Fibrous
Pinkish
79.32 ± 1.70a
17.19 ± 0.54a
MAIS1
Cottony
Whitish to greyish
60.44 ± 4.63b
13.47 ± 0.89b
MAIS2
Compact
Greyish green
40.06 ± 4.17c
7.37 ± 0.72c
PAIS1
Cottony
Whitish to greyish
65.27 ± 3.84b
13.41 ± 0.77b
PLIS1
Cottony
Whitish to greyish
54.05 ± 3.51b
13.14 ± 0.60b
‫٭‬Means in columns followed by the same letter are not significantly
level.
Morphological characteristics
Conidia produced by isolates AVIS1, BAIS1, MAIS1, PAIS1
and PLIS1 varied from fusiform with obtuse to slightly
rounded ends to sometimes oblong. BAIS2 produced
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different by Duncan’s Multiple Range test at a 5% probability
cylindrical conidia with obtuse to slightly rounded ends.
AVIS2 and MAIS2 produced fusiform conidia with obtuse
ends (oblong) with narrowing at the centre (Fig 2)
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Vol-1, Issue-4, Nov-Dec- 2016
ISSN: 2456-1878
Fig.2: Conidia morphology of Colletotricum spp. isolated from various fruits. (a) AVIS1, BAIS1,
MAIS1, PAIS1 and PLIS1; (b) BAIS2; (c) AVIS2 and MAIS2.
x 106 to 9.42 x 106 conidia/ml.AVIS2 and MAIS2 produced
have cylindrical conidia of smaller sizes which vary from
Conidia produced by BAIS2 had biggest sizes (25.62 x 6.04
15.72 x 3.14 to 16.43 x 3.52 µm as well as sporulation rates
µm) and had the most abundant sporulation rate on the 10th
6
days (13.19 x 10 conidia/ml). AVIS1, BAIS1, MAIS1, PAIS1
which varied from 7.21 x 106 to 7.26 x 106 conidia/ml (Table
and PLIS1 produced conidia which and measured 19.98 –
3).
21.29 x 4.17 – 5.14 µmwith sporulation rates ranging from 7.7
Table.3: Conidia shape, size and sporulation rate of Colletotrichum isolated from different fruits.
Isolate
Conidial shape
Size of conidia (µm)
Sporulation rate on day 10 (x 106 conidia/ml)
Fusiform
20.71 x 4.65
9.42 ± 0.43b
AVIS1
BAIS1
Fusiform
21.29 x 5.14
8.27 ± 0.62b
MAIS1
Fusiform
19.91 x 4.17
7.50 ± 0.61bc
PAIS1
Fusiform
19.98 x 4.89
7.81 ± 0.97bc
PLIS1
BAIS2
AVIS2
Fusiform
Cylindrical
Fusiform
20.43 x 4.77
25.62 x 6.04
15.72 x 3.14
7.70 ± 0.80bc
13.19 ± 0.47a
7.21 ± 0.74c
Fusiform
16.43 x 3.52
7.26 ± 0.94c
MAIS2
‫٭‬Means in columns followed by the same letter are not significantly different by Duncan’s Multiple Range test 5% at a probability
level.
Dendrogram resulting from the hierarchical cluster analysis of
mycelial growth rate, diameter of colonies and size of conidia
of Colletotrichum spp isolates from various fruits showed three
differents groups; Group I (MAIS1, PAIS1, PLIS1, BAIS1 and
AVIS1), group II (AVIS2 et MAIS2) and group III (BAIS2)
(Fig 3).
Fig.3: Dendrogram resulting from the hierarchical cluster
analysis showing the groups formed according to the
variables; mycelial growth rate, diameter of colonies and size
of conidia after seven days cultivation of Colletotrichum spp
from various fruits.
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IV.
DISCUSSION
The results of the study indicate that Colletotrichum species
isolated from fruits of avocado, banana, mango, pawpaw and
plums show some variations in cultural and morphological
characters. Based on these characteristics, Colletotrichum
isolates were ranked into three groups; the first group made up
of AVIS1, MAIS1, BAIS1, PAIS1 and PLIS1 isolates with
whitish to greyish colonies and fusiform conidia, the 2nd group
made up AVIS2 and MAIS2 with respectively fibrous and
compact greyish green mycelia colonies and cylindrical
conidia and the 3rd group (BAIS2) with pinkish mycelia and
cylindrical conidia. The difference in coloration and the aspect
of the isolates would be related to the fruit host, the nature of
the Colletotrichum isolate and the environmental conditions.
Identification of Colletotrichum species was reported to be
mostly based on morphological and cultural criteria, coupled
with knowledge of the host origin of the pathogen. However,
many isolates of Colletotrichum show extensive variation in
culture (Sutton, 1992; Bailey et al., 1995). Several cultural and
morphological types have been observed on Colletotrichum
species isolated from fruits of mango (Sanders and Korsten,
2003; N’Guettia et al., 2013), bananas (Cannon et al., 2008;
Prihastuti et al., 2009; Su et al., 2011), pawpaw (Rampersad,
2011) and avocados (Keuete, 2014).
The mycelial growth differentiated three groups of isolates 7
days after culture. The 1st group (BAIS2) recorded a fast
growth (17.19 mm/day), 2nd group (AVIS1, BAIS1, MAIS1,
PAIS1 and PLIS1) recorded an average growth of 13.02 to
13.62 mm/day and the 3rd group (AVIS2 and MAIS2) recorded
a slow growth (7.37 to 7.62 mm/day). The mycelial growth has
been reported to be a criterion that helps to differentiate the
species of Colletotrichum (Waller et al., 1993; Crouch et al.,
2009; Liu et al., 2012). However, according to Serra et al.
(2006).Although it is not a stable criterion of differentiation of
Colletotrichum species, but it plays a significant role in
variability within the species. Bailey et al. (1995) argued that
many isolates of Colletotrichum often show extensive variation
in culture and furthermore, the culture conditions , including
the media,the age of culture and the temperatures used, cannot
be standard betweenlaboratories (Sutton 1992).
Conidial morphology has always been emphasized over other
taxonomic criteria in taxonomic investigations of the genus
Colletotrichum. Baxter et al. (1983) used conidial shape and
size as the main criteria to distinguish a number of species.
According to Sutton (1992) and Bailey et al. (1995),
identification of Colletotrichum species had been mostly based
on these criteria, coupled with knowledge of the host origin of
the pathogen, however, extensive variation of many isolates of
Colletotrichum had been shown in culture.
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Vol-1, Issue-4, Nov-Dec- 2016
ISSN: 2456-1878
On the basis of conidia size and shape, Colletotrichum isolates
were divided into three groups. The first also producing
fusiform shapes conidia whose sizes varied from 19.98 x 4.17
to 21.29 x 5.14 µm. These values fall within the interval
described by Rivera et al. (2006) for conidia of C.
gloeosporioides. C. gloeosporioides had been considered to be
a group species or species complex found on a wide variety of
fruits, such as apple, avocado, citrus, papaya, peach, mango
and strawberry (Freeman 2000). The variation may also be
attributed to the adaptation of the species to a non-specific,
broad host range (Freeman et al. 1998). For Colletotrichum
species, it is common for single hosts to become infected by a
single species or for multiple hosts to be infected by a single
species of the pathogen (Freeman 2000). Infection of multiple
hosts by C. musae has been reported by Su et al. (2011).
The 2nd group consistingof AVIS2 and MAIS2 isolates was
characterised by fusiform conidia with the sizes lying between
15.72 x 3.14 and 16.43 x 3.52 µm.Freeman (2002), Than et al.
(2008), Peres et al. (2008) and Damm et al. (2012) observed
similar shape and size of conidia on C. acutatum. The 3rd group
made up of BAIS2 isolates had spindle-shaped conidia with the
sizes of 25.62 x 6.04 µm. The grouping of Colletotrichum
isolates into three subclades suggest that the isolates may
represent a sub-population of the pathogens with distinct
genetic characters. Similar results were reported by Prihastuti
et al. (2009) and Waller et al. (1993) in which C.
gloeosporioides on coffee berries showed several distinct
genetic and phenotypic species. However, it is not yet known,
whether these isolates are distinct or not.
V.
CONCLUSION
The study highlights some variation in morphocultural
characterisrics of Colletotrichum species from various fruits,
but sequence analysis are still to be carried on to confirm the
existence of more than one distinct isolates since
Colletotrichum has wide host range.
ACKNOWLEDGEMENTS
The authors are thankful to Phytopathology Laboratory,
University of Dschang, Cameroon and for Providing
Laboratory Facilities and grateful to Dr. M. Lekefack for the
proofreading of the manuscript.
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